1. Field of the Invention
In recent years, there has been a growing recognition that enzymes can be used effectively in analytical, medical, and industrial applications. Enzymes are biological catalysts and all known enzymes are proteins having a relatively high molecular weight. As catalysts, enzymes can promote various chemical reactions which utilize specific enzymes or enzyme systems. Typically, an enzyme system acts on one or more compounds designated substrates to produce one or more products.
Enzymes have been classified into various groups depending on the reactions they promote and the substrates they can act upon. These groups of enzymes (e.g., hydrolytic enzymes, redox enzymes, transferase enzymes) are known in the art and they can be conveniently broken down into sub-groups which are commonly descriptive of the substrate the enzyme can act upon (e.g., redox enzymes are those that catalyze oxidation or reduction reactions and which include, for example, the enzyme glucose oxidase which promotes the oxidation of glucose).
Enzymes are normally soluble in an aqueous solution. This solubility has made it difficult to readily remove the enzymes from a solution for repeated use and/or to maintain the catalytic activity of the enzymes for extended periods of time. For those reasons, and because of the growing use of enzymes, various methods have been developed to insolubilize or immobilize the normally soluble enzymes in such manners that the enzymes retain their activity, can be readily removed from a reaction solution, and used repeatedly.
Enzymes have been insolubilized and immobilized in a variety of ways. For example, enzyme composites have been made by physyically entrapping enzymes in such organic materials as starch gel, polyacrylamide gel, agar and the like. They have also been insolubilized by chemically coupling them via azo linkage to cellulose derivatives and to polyaminostyrene beads. Enzymes have also been insolubilized on polytyrosyl polypeptides and in colloidion matrices.
Several disadvantages have been associated with using the above organic materials. The organic materials have been found subject to microbial attack resulting from the presence of carbon atoms in the polymer chain whereby the carrier is broken down and the enzymes become solubilized. Also, many organic carriers have poor heat stability and thus, are difficult to sterilize by such means as an autoclave. Further, some organic materials lack dimensional stability when used in columns and an increase or decrease in swelling of the materials can affect the flow rates of a substrate flowing through the column or the conformation of an attached enzyme. The above disadvantages, and others, associated with using organic carriers or organic matrices for insolubilizing or immobilizing enzymes led to the development of enzyme composites utilizing inorganic carriers. The present invention represents an improvement in the field of enzyme composites which utilize inorganic carriers.
2. Prior Art
In U.S. Pat. No. 3,556,945, assigned to the assignee of the present invention, there are disclosed enzyme composites in which the enzyme is adsorbed to an essentially insoluble inorganic carrier such as porous glass. In U.S. Pat. No. 3,519,538, also assigned to the same assignee, there are disclosed enzyme composites which consist of inorganic carriers to which enzymes are chemically coupled via intermediate silane coupling agents. In the above patent, it is pointed out that by chemically coupling the enzymes to an inorganic carrier, the problems associated with organic carriers are avoided as well as the limitations associated with adsorbing enzymes to inorganic carriers, e.g., adsorption of enzymes to inorganic carriers results in a relatively weak bond (compared to covalent coupling) and it is not general for all enzymes since a loss of activity results when there is adsorptive bonding at the active sites on the enzyme molecule. The present invention is an improvement of the inventions disclosed in the above enzyme coupling patent and the teachings of that patent are incorporated herein by reference to that patent (U.S. Pat. No. 3,519,538).
In the above patent it was disclosed that chemically coupled enzymes retained their activity for considerably greater time periods than the corresponding soluble, noncoupled enzyme. However, even though the chemically coupled enzyme composites were found to retain their activity much longer than the free, soluble enzymes, there have been certain disadvantages associated with the coupled enzyme composites. The main disadvantage relates to the half-life of the composites. The term half-life, as used herein, refers to the period of time which elapses before the composite loses one-half of its original enzymatic activity. Enzymatic activity is commonly measured in activity units. Each unit refers to an amount of product which can be produced through the catalytic action of an enzyme over a given time period at a given temperature and pH. For example, the activity of the enzyme glucose isomerase (which converts glucose to fructose), when expressed in terms of International Units (IU), can be expressed as follows:
One unit (IU) = the production (or capability of production) of 1 .mu. mole fructose per minute at 60.degree. C., pH 6.85. Also, the activity of the enzyme glucoamylase (which converts starch to dextrose) is commonly expressed in terms of International Units (although not accepted officially) such that one unit = the production (or capability of production) of 1 .mu. mole dextrose per minute at 60.degree. C., pH 4.5. An enzyme system can be assigned an operational half-life, based on activity loss with time or a calculated half-life based on initial and final activity.
Even though chemically coupled enzymes have demonstrated half-lives longer than free enzymes, such half-lives have generally been measurable in hours, days, or weeks rather than months or years. This relatively limited half-life of coupled enzymes has greatly limited their utility. This is especially true in industry, since it has been found in some cases that the cost of preparing and using chemically coupled enzyme composites of limited half-life may not generally result in significant cost savings over conventional usage of the free, soluble enzyme.
If an immobilized enzyme composite is to compete economically with the corresponding soluble enzyme, its cost of manufacture and usage must be at least as low as the cost and usage of the soluble enzymes needed to produce a given amount of product. The value of an insolubilized enzyme composite is generally related directly to its half-life and the amount of enzyme which can be loaded on a given amount of carrier. Thus, if an enzyme composite has a half-life such that the cost of its manufacture and use to produce a given amount of product is less than the cost of using the free enzyme to produce the same amount of product, then the value of the enzyme composite becomes greater than the value of the free enzyme. Accordingly, it then becomes more feasible to use the insoluble enzyme composite. For this reason, considerable attention has been directed toward discovering methods for increasing the half-life of enzyme composites having inorganic carriers.
We have now discovered methods for preparing an insolubilized enzyme composite which has a significantly longer half-life than previously obtained. Our discovery was quite surprising since we found the half-life of insoluble enzyme composites having an inorganic carrier is not as much attributable to the nature of the enzyme itself or the enzyme coupling procedure as it is to the inorganic carriers used in the coupled enzyme composite.